Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds

Abstract

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

Genetic Analysis of Six Transmembrane Protein Family Genes in Parkinson’s Disease in a Large Chinese Cohort

“…Our study identified several rare non-synonymous TMEM175 , TMEM163 , and TMEM229B variants, but no association of these genes with PD was found. TMEM175 is a component of a lysosomal K + channel, and its deficiency results in impaired lysosomal and mitochondrial function ( Jinn et al, 2017 ; Pergel et al, 2021 ). Variants of TMEM175 have been identified to be associated with PD in a GWAS study, indicating its role in PD ( Nalls et al, 2014 ).…”

Transmembrane Protein 175, a Lysosomal Ion Channel Related to Parkinson’s Disease

“…TMEM175 currents in the plasma membrane were greatly increased in the cells treated with dynasore, with an estimated EC 50 of at least 30 µM [88]. Dyngo-4a, a hydroxylated derivative of dynasore, induces more pronounced TMEM175 currents with an estimated EC 50 of 2.3 µM [88]. Following the removal of dynasore chemicals, TMEM175 currents rapidly decreased, indicating the utilization of an effective internalization mechanism to translocate TMEM175 proteins from the plasma membrane to intracellular organelles.…”

“…Additionally, the heterologous expression of mouse TMEM175 in Xenopus laevis oocytes results in very small currents through the plasma membrane of oocytes. Dynasore compounds, which are potent dynamin inhibitors and disrupt clathrin-dependent endocytosis, have been reported to control the cellular localization of TMEM175 proteins [88]. TMEM175 currents in the plasma membrane were greatly increased in the cells treated with dynasore, with an estimated EC 50 of at least 30 µM [88].…”

“…Dynasore compounds, which are potent dynamin inhibitors and disrupt clathrin-dependent endocytosis, have been reported to control the cellular localization of TMEM175 proteins [88]. TMEM175 currents in the plasma membrane were greatly increased in the cells treated with dynasore, with an estimated EC 50 of at least 30 µM [88]. Dyngo-4a, a hydroxylated derivative of dynasore, induces more pronounced TMEM175 currents with an estimated EC 50 of 2.3 µM [88].…”

Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters

“…Furthermore, surface expression of intracellular transport proteins upon manipulation of sorting/internalization signals represents a powerful tool for their functional characterization, which in some cases even was the only successful approach so far. There is also one report, in which the use of inhibitors of dynamin redirected the lysosomal K + channel c to the plasma membrane upon expression in Xenopus oocytes [112].…”